Metal Resistant Plants, and Methods of Manufacture Thereof

ABSTRACT

Disclosed herein is a transgenic plant transformed with an isolated nucleic acid comprising a plant arsenite-inducible RNA-associated protein coding sequence operatively linked to a plant-expressible transcription regulatory sequence, wherein the plant arsenite-inducible RNA-associated protein (AIRAP) coding sequence encodes a polypeptide that is at least 95% identical to a polypeptide sequence of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, wherein the plant arsenite-inducible RNA-associated protein coding sequence encodes a polypeptide that confers resistance to an environmental stress, wherein greater than or equal to about 25% of transgenic plants are resistant to an environmental stress, and wherein the environmental stress inhibits the growth of wild type plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 12/125,362, filed May 22, 2008, which claims priority to U.S.Provisional Patent Application Ser. No. 60/939,751, filed May 23, 2007,all of which are incorporated by reference herein in its entirety.

BACKGROUND

Abiotic stresses such as drought, salt, nutrient deficiency, andexposure to toxic metals adversely affect the growth and productivity ofcrop plants and thus are serious threats to global agriculturalproduction and food security. These environmental stresses reduces cropyield by more than 30 to 50%, which not only cause the loss of billionsof dollars annually, but also threaten the sustainability of the globalagricultural industry.

Metal and metalloid pollutants such as arsenic (As), cadmium (Cd),chromium (Cr), lead (Pb), mercury (Hg), and zinc (Zn), can adverselyaffect the health of millions of people worldwide. Arsenic, for example,is toxic and carcinogenic. The metal and metalloid contaminated soil,sediment, and water supplies are major sources of contamination in thefood chain. Metal and metalloid poisoning can occur via ingestion ofcontaminated drinking water and food. Industrial pollution andagricultural practices including the use of metal andmetalloid-containing pesticides, herbicides, fertilizers, and woodpreservatives, as well as irrigation with contaminated groundwater, andmining have significantly increased metal and metalloid contamination inagricultural soil. There is global concern regarding arseniccontamination in drinking water and soil, particularly on the Indiansubcontinent where more than 450 million people are at risk for arsenicpoisoning.

There are many different ways metal and metalloid pollutants can enterthe food chain. Plants grown in contaminated soil can accumulate highlevels of metal and metalloid pollutants in roots, shoots, and grain.Metal and metalloid pollutant uptake by plants may play an importantrole in the introduction of these pollutants into the food chain, forexample, by the direct ingestion of contaminated grain. In addition,contaminated straw that is used as cattle feed may have adverse healtheffects on cattle and may result in increased metal and metalloidexposure in humans via a plant-animal-human pathway. There is,therefore, concern regarding the accumulation of metal and metalloidpollutants in meat and dairy products as well as in agricultural cropsand vegetables.

In addition, metal and metalloid pollutants are phytotoxic and causesignificant loss in crop yields. For example, arsenate is a phosphateanalog and competes with phosphate for uptake in plants causing theinhibition of phosphate and other nutrients. Thus, arsenic contaminationis an agricultural concern. A plant that is resistant to metal andmetalloid pollutants and can accumulate a large biomass despite thepresence of metal and metalloid pollutants will be advantageous as abiofuel plant. Such a plant could be grown on contaminated, butotherwise arable, land.

Metals and metalloids are often present in the environment in differentionic forms. With respect to arsenic, the arsenate oxyanions, HAsO₄ ²⁻and H₂AsO₄ ⁻, are the most prevalent forms of arsenic in surface soil,water, and within cells, and these oxyanions contain arsenic in thepentavalent state [As(V)]. Arsenite, which at neutral pH containsarsenic in the trivalent oxidation state [As(III)] and likely as theacid HAsO₃ ²⁻, is highly reactive and readily forms As(III)-thiolcomplexes. Plants use arsenate reductases to detoxify arsenic byreducing As(V) to As(III), which is subsequently detoxified via formingcomplexes with thiol-reactive peptides such as γ-glutamylcysteine(γ-EC), glutathione (GSH) and phytochelatins (PCs). It is suggested thatthese As(III)-thiol complexes are then sequestered into vacuoles byglutathione-conjugating pumps. It is further believed that plants traparsenite in below ground tissues in order to prevent access to aboveground reproductive tissues to prevent possible mutagenic consequences.

Similarly, another environmental factors, such as salinity, heat, cold,drought, and floods can limit the amount of arable land for cropproduction as well as reduce crop yields. Developing crops with enhancedabiotic stress tolerance will undoubtedly have an important effect onglobal food production and will help to alleviate an increasinglyimminent threat of famine.

Accordingly, there is a need to develop novel plants that are resistantto environmental or abiotic stresses.

SUMMARY

Disclosed herein is a transgenic plant transformed with an isolatednucleic acid comprising a plant arsenite-inducible RNA-associatedprotein coding sequence operatively linked to a plant-expressibletranscription regulatory sequence, wherein the plant arsenite-inducibleRNA-associated protein (AIRAP) coding sequence encodes a polypeptidethat is at least 95% identical to a polypeptide sequence of SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, orSEQ ID NO:14, wherein the plant arsenite-inducible RNA-associatedprotein coding sequence encodes a polypeptide that confers resistance toan environmental stress, wherein greater than or equal to about 25% oftransgenic plants are resistant to an environmental stress, and whereinthe environmental stress inhibits the growth of wild type plants.

Further disclosed is a method for producing a transgenic plant that isresistant to an environmental stress comprising introducing an isolatednucleic acid comprising an plant arsenite-inducible RNA-associatedprotein coding sequence that encodes a polypeptide that is at least 95%identical to a polypeptide sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, and SEQ ID NO:14 operatively linked to a plant-expressibletranscription regulatory sequence into a plant cell or plant tissue;producing a transgenic plant cell or tissue comprising the isolatednucleic acid; and regenerating the transgenic plant cell or transgenicplant tissue to provide a transgenic plant that is resistant to anenvironmental stress, wherein greater than or equal to about 25% oftransgenic plants are resistant to the environmental stress, and whereinthe environmental stress inhibits the growth of wild type plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of the following plant SAP proteins:AtAIRAP1 (new name AtSAP11) (SEQ ID NO:8); AtAIRAP2 (new name AtSAP13)(SEQ ID NO:9); AtAIRAP3 (new name AtSAP14) (SEQ ID NO:10); AtAIRAP4 (newname AtSAP12) (SEQ ID NO:11); AtAIRAP5 (new name AtSAP10) (SEQ IDNO:12); OsAIRAP1 (new name OsSAP16) (SEQ ID NO:13); and OsAIRAP2 (newname OsSAP17) (SEQ ID NO:14);

FIG. 2 shows a schematic diagram of a cloning strategy of arepresentative vector construct used to transform plants;

FIG. 3 shows PCR amplification of Arabidopsis and rice SAP cDNAs;

FIG. 4 shows PCR amplification of Arabidopsis and rice SAP cDNAs aftermetal or metalloid induction;

FIG. 5 shows PCR amplification of AtSAP13 cDNA from several transgenicArabidopsis lines;

FIG. 6 shows semi-quantitative RT-PCR analysis of AtSAP10 expression inresponse to various stress treatments;

FIG. 7 shows a transgenic Arabidopsis line that is resistant to severalmetals and metalloids;

FIG. 8 shows a transgenic Arabidopsis line that is resistant to nickel;

FIG. 9 shows a transgenic Arabidopsis line that is resistant tomanganese;

FIG. 10 shows a transgenic Arabidopsis line that is resistant to zinc;

FIG. 11 shows metal and metalloid accumulation in a transgenicArabidopsis line;

FIG. 12 shows metal and metalloid accumulation in a transgenicArabidopsis line;

FIG. 13 shows salt resistance in a transgenic Arabidopsis line;

FIG. 14 shows heat resistance in a transgenic Arabidopsis line; and

FIG. 15 shows drought resistance in a transgenic Arabidopsis line.

DETAILED DESCRIPTION

Disclosed herein is an isolated nucleic acid comprising a sequence atleast 95% identical to the nucleotide sequence of AtSAP11 (GenBank No.At2g41835; SEQ ID NO:1); AtSAP13 (GenBank No. At3g57480; SEQ ID NO:2);AtSAP14 (GenBank No. At5g48205; SEQ ID NO:3); AtSAP12 (GenBank No.At3g28210; SEQ ID NO:4); AtSAP10 (GenBank No. At4g25380; SEQ ID NO:5);OsSAP16 (GenBank No. Os09g38240.1; SEQ ID NO:6); or OsSAP17 (GenBank No.Os09g21710.1; SEQ ID NO:7). In another embodiment, the isolated nucleicacid encodes a polypeptide that confers resistance to an environmentalstress when expressed in a plant.

Disclosed herein also is an isolated nucleic acid comprising a sequencethat encodes a polypeptide that is at least 95% identical to the aminoacid sequence of AtSAP11 (GenBank No. At2g41835; SEQ ID NO:8); AtSAP13(GenBank No. At3g57480; SEQ ID NO:9); AtSAP14 (GenBank No. At5g48205;SEQ ID NO:10); AtSAP12 (GenBank No. At3g28210; SEQ ID NO:11); AtSAP10(GenBank No. At4g25380; SEQ ID NO:12); OsSAP16 (GenBank No.Os09g38240.1; SEQ ID NO:13); or OsSAP17 (GenBank No. Os09g21710.1; SEQID NO:14). In another embodiment, the isolated nucleic acid encodes apolypeptide that confers resistance to an environmental stress.

Disclosed herein is an environmental stress resistant transgenic plantand methods of manufacture thereof. In one embodiment, provided is atransgenic plant transformed with an isolated nucleic acid, the isolatednucleic acid comprising a plant SAP coding sequence operatively linkedto a plant-expressible transcription regulatory sequence. The inventorshave discovered that the increased expression of a plant SAP gene in atransgenic plant dramatically increases the environmental stressresistance of the transgenic plants. An additional advantage is that theincrease in environmental stress resistance also results in increasedbiomass of the transgenic plants. The environmental or abiotic stressesinclude environmental stresses, such as, but not limited to, toxicmetals, high salt concentrations, drought, cold, heat, and submergence.

In one embodiment, the transgenic plants further comprise an isolatednucleic acid suitable for expression of a coding sequence for an enzymeinvolved in the biosynthesis of the phytochelatins. Phytochelatins arepeptides of higher plants having a general structure of(γ-Glu-Cys)_(n)-Gly, where n equals 2 to 11. Phytochelatins aresynthesized in plants in response to the presence of heavy metals andform stable complexes with metal ions. Exemplary phytochelatinbiosynthetic enzymes include γ-ECS (γ-glutamylcysteine synthase), GS(glutathione synthase), and PCS (phytochelatin synthase). In oneembodiment, both isolated SAP and phytochelatin biosynthetic enzymerecombinant genes (transgenes) are combined in a single plant genome bycotransformation of two constructs, by sequential transformation, or bycross-breeding singly transformed plants, each containing one of thegenetic constructs of interest, with selection for progeny having boththe SAP coding sequence and the phytochelatin biosynthetic codingsequence. In another embodiment, two or more transgenes are combined ina single plant by conventional breeding and screening (phenotypic or formolecular markers) to obtain the plant that express both the recombinantSAP gene and the recombinant phytochelatin biosynthetic enzyme gene.

In animals, it has been shown that AIRAPs are selectively induced inresponse to As(III) (Sok et al., 2001). AIRAP mRNA was induced more than15-fold by As(III) treatment and 5-fold by Zn treatment in mouseepithelial cells. The mouse AIRAP homologous sequences were identifiedin Caenorhabditis elegans, Drosophila melanogaster, and human (Sok etal., 2001). The amino acid sequence alignment of these related proteinsrevealed the presence of highly conserved motifs with 8 cysteines andhistidines repeated twice in the protein. The arrangement of cysteineand histidine residues in these AIRAPs is similar to those known to formmetal coordination complex similar to the RING-finger type (Saurin etal., 1996) but does not conform to the strict RING or RING-H2 consensus.These proteins have been shown to protect Caenorhabditis elegans andhuman cells from As(III) toxicity but their exact function is not known.The RNAi knockdown of C. elegans homologue of AIRAP, aip-1, lowers theresistance of nematodes to As(III) but did not affect viability withoutAs(III) exposure (Sok et al., 2001)) Immunoprecipitation and cellfractionation experiments in mouse cell indicated that, when induced,AIRAP is present in both the nucleus and the cytoplasm. Further, in vivocross-linking experiments indicated that AIRAP is associated with RNA(Sok et al., 2002), and hence their name RNA-associated proteins. Theseresults indicate that AIRAP functions in association with RNA, however,their exact function remain unknown so far.

The inventors have discovered plant homologues of AIRAP. The Arabidopsishomologues (AtSAP10-14) encode for polypeptides corresponding to 130,279, 186, 249, and 191 amino acid residues, respectively. The AtAIRAPhomologous sequences are also conserved in other plant species such asrice and brassica. The rice homologues (OsSAP16-17) encode forpolypeptides corresponding to 290 and 188 amino acid residues,respectively.

The inventors identified a family of 14 and 18 AIRAP homologous proteinsin Arabidopsis and rice, respectively. These genes encode hypotheticalproteins containing A20/AN1 zinc-fingers with unknown functions.Recently, the members of this A20/AN1 type zinc finger protein familywere designated as Stress Associated Proteins, SAPs (Vij and Tyagi,2006). Accordingly, the Arabidopsis and rice genes are currentlyreferred to as AtSAPs and OsSAPs, respectively. Table 1 shows the newSAP gene names and the corresponding old AIRAP gene names.

TABLE 1 Seq ID No: Seq ID No: Gene name (DNA) (protein) (Old) Locus IDNew name 1 8 AtAIRAP1 At2g41835 AtSAP11 2 9 AtAIRAP2 At3g57480 AtSAP13 310 AtAIRAP3 At5g48205 AtSAP14 4 11 AtAIRAP4 At3g28210 AtSAP12 5 12AtAIRAP5 At4g25380 AtSAP10 6 13 OsAIRAP1 Os07g38240.1 OsSAP16 7 14OsAIRAP2 Os0921710.1 OsSAP17

In Arabidopsis and rice genomes, there are 14 and 18, respectively,A20/AN1 zinc-finger type proteins including the SAP homologues (Jin etal., 2007; Vij and Tyagi, 2006). Out of the 14 reported Arabidopsisproteins, 10 are reported to contain A20-AN1 type zinc finger domainsand four as AN1 type zinc-finger domains. The Arabidopsis AtSAP10-14 andrice OsSAP16-17 proteins disclosed herein are AN1 type zinc fingerproteins and contain Cys2-His2 finger motifs. Based on a phylogeneticanalysis of the AN1 zinc finger domain, Jin et al., (2007) recentlydivided all A20/AN1 zinc finger genes into two groups, Type I and TypeII. Type I gene contains the traditional pattern,CX₂CX_(9,12)CX_(1,2)CX₄CX₂HX₅HXC, whereas, Type II contains the expandeddomain, CX₄CX₂CX_(9,12)CX_(1,2)CX₄CX₂HX₅HXC where X represents any aminoacid. There are ten members from Arabidopsis and fifteen members fromrice in the Type I genes group. The Type II genes group includes ninemembers—three from Arabidopsis and two from rice. Type I genes containone intact A20 type and one AN1 type zinc finger domain, whereas, TypeII genes contain two intact AN1 type zinc finger domain and lack A20type domain (Jin et al., 2007). Based on the phylogenetic analysis ofType II genes, Jin et al. (2007), further divided Type II group into twosubclasses—class IIA and class IIB. Class IIA members, in addition toAN1 domain, also contain extra C₂H₂ type zinc finger domain atC-terminal. Two Arabidopsis SAPs (AtSAP11 and AtSAP13) and the riceOsSAP16 belong to class IIA. The third and fourth Arabidopsis proteinAtAIRP3 and AtSAP12 represent class IIB because they lack the extraC-terminal C₂H₂ type zinc finger domain. There is a striking differencein the structure of A20/AN1 zinc finger genes in plants and animals.Only two human Type I genes have protein structure similar to those ofplants and all human/animal Type II genes have protein structuresdifferent from those of plants. This led Jin et al. (2007) to concludethat there is a plant-specific protein structure for Type II genes.

The Arabidopsis SAP protein sequences are highly cysteine andhistidine-rich and have more than 60% similarity to the animal proteinin the conserved sulfur-rich region proposed to bind As(III). As shownin FIG. 1, alignment of the predicted protein sequences of the fiveArabidopsis SAPs reveal the presence of highly conserved cysteine andhistidine repeats arranged in particular configurations. (FIG. 1) Forexample, the AtSAP11, AtSAP13, AtSAP14, AtSAP12, AtSAP10 sequencescontain a distinct pattern of 19, 18, 9, 16, and 12 conserved cysteineresidues, respectively.

The inventors have further unexpectedly discovered that plants, such asArabidopsis thaliana, genetically engineered to overexpress a plant SAPgene, demonstrate improved metal resistance. Without being bound bytheory, it is hypothesized that the conserved Cys2-His2 zinc fingerdomains are involved in coordinating and binding metals. According tothis model, plants have improved metal resistance because the metals aresequestered in these zinc finger-metal complexes. Similar results areobtainable in other plants, including monocots, dicots and gymnosperms,after stable transformation and regeneration. Suitable plants alsoinclude field crops, fruits, and vegetables such as canola, sunflower,tobacco, mustard, crambe, sugar beet, cotton, maize, wheat, barley,rice, sorghum, mangel-wurzels, tomato, mango, peach, apple, pear,strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion,soybean, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa,rye, oats, turf and forage grasses, flax and oilseed rape, nut producingplants, and the like. Suitable plants also include biofuel, biomass, andbioenergy crop plants. Exemplary plants include Arabidopsis thaliana,rice (Oryza sativa), switchgrass (Panicum vigratum), Brachypodium spp,corn (Zea mays), Sorghum spp, barley (Hordeum vulgare), wheat (Triticumaestivum), Brassica spp., and Crambe abyssinica.

As used herein, the term “metal resistance” means that a non-naturallyoccurring organism (e.g., a transgenic plant) is not inhibited by thepresence of at least one ionic form of a metal or metalloid at aconcentration or amount that inhibits or is toxic to a naturallyoccurring (wild type) counterpart of the non-naturally occurringorganism. It is not intended that the term metal resistance refer toresistance to unlimited metal concentrations, but rather the term isrelative in that it relies on comparison to the properties of a parentalstrain. “Metal” refers to an element classified as a metal or metalloidas well any ionic forms of the metal or metalloid elements. Exemplarymetals include arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb),mercury (Hg), nickel (Ni), manganese (Mn), iron (Fe), and zinc (Zn). Inone embodiment, a metal resistant organism is resistant to a metalconcentration of greater than or equal to about 50 micromolar.Specifically, a metal resistant organism is resistant to a metalconcentration of greater than or equal to about 100 micromolar. Morespecifically, a metal resistant organism is resistant to a metalconcentration of greater than or equal to about 500 micromolar. Evenmore specifically, a metal resistant organism is resistant to a metalconcentration of greater than or equal to about 1 millimolar.

In one embodiment, the metal resistant transgenic plant is alsoresistant to other environmental stresses, including, but not limited toabiotic stresses such as high salt concentration, drought, cold, andsubmergence. As used herein, the term “stress resistance” means that anon-naturally occurring organism (e.g., a transgenic plant) is notinhibited by an environmental stress that inhibits or is toxic to anaturally occurring (wild type) counterpart of the non-naturallyoccurring organism. It is not intended that the term stress resistancerefer to resistance to unlimited stress (e.g., concentration,temperature, or duration), but rather the term is relative in that itrelies on comparison to the properties of a parental strain. Stressrefers to environmental conditions such as high salt concentration,drought, cold, and submergence that inhibit growth or are toxic to awild type plant. In one embodiment, a stress resistant organism isresistant to a one-week exposure to a salt concentration of greater thanor equal to about 50 millimolar. Specifically, a stress resistantorganism is resistant to a one-week exposure to a salt concentration ofgreater than or equal to about 100 millimolar. More specifically, astress resistant organism is resistant to a one-week exposure to a saltconcentration of greater than or equal to about 250 millimolar. Evenmore specifically, a stress resistant organism is resistant to aone-week exposure to a salt concentration of greater than or equal toabout 500 millimolar.

An “SAP sequence” is one that encodes a protein capable of mediatingresistance to metals or metalloids and their ions, including, but notlimited to, arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb),mercury (Hg), nickel (Ni), manganese (Mn), and zinc (Zn). Also withinthe scope of this definition are variant sequences that encode proteinscapable of mediating resistance to metals or metalloids and their ions.Suitable SAP sequences include the Arabidopsis thaliana SAP1-5 sequencesand the rice SAP1-2 sequences.

In one embodiment, included herein are isolated SAP nucleic acids. Anisolated nucleic acid is a nucleic acid the structure of which is notidentical to that of any naturally occurring nucleic acid or to that ofany fragment of a naturally occurring genomic nucleic acid spanning morethan three separate genes. The term therefore covers, for example, (a) aDNA that has the sequence of part of a naturally occurring genomic DNAmolecule but is not flanked by both of the coding or noncoding sequencesthat flank that part of the molecule in the genome of the organism inthat it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of (i) DNA molecules, (ii) transformed or transfected cells,and (iii) cell clones, e.g., as these occur in a DNA library such as acDNA or genomic DNA library.

In one embodiment, the SAP comprises the Arabidopsis thaliana AtSAP11sequence (nucleotide sequence SEQ ID NO:1, Accession numberNM_(—)180027.1; polypeptide sequence SEQ ID NO:8, Accession numberNP_(—)850358.1). In another embodiment, the SAP comprises the AtSAP13sequence (nucleotide sequence SEQ ID NO:2, NM_(—)115608.3; polypeptidesequence SEQ ID NO:9, Accession number NP_(—)191307.1). In anotherembodiment, the SAP comprises the AtSAP14 sequence (nucleotide sequenceSEQ ID NO:3, Accession number NM_(—)203175.1; polypeptide sequence SEQID NO:10, Accession number NP_(—)974904.1). In another embodiment, theSAP comprises the AtSAP12 sequence (nucleotide sequence SEQ ID NO:4,Accession number NM_(—)113740.4; polypeptide sequence SEQ ID NO:11,Accession number NP_(—)189461.1.) In another embodiment, the SAPcomprises the AtSAP10 sequence (nucleotide sequence SEQ ID NO:5;Accession number NM_(—)118670.1; polypeptide sequence SEQ ID NO:12;Accession number NP_(—)194268.1.) In another embodiment, the SAPcomprises the OsSAP16 sequence (nucleotide sequence SEQ ID NO:6;Accession number gi|133146764; polypeptide sequence SEQ ID NO:13;Accession number gi|133146779.) In another embodiment, the SAP comprisesthe OsSAP16 sequence (nucleotide sequence SEQ ID NO:7; Accession numbergi|149387714; polypeptide sequence SEQ ID NO:14; Accession numbergi|140363792.)

An SAP includes an SAP homologous to AtSAP10-14 or OsSAP16-17 so long asthe SAP has SAP activity. “Homolog” is a generic term used in the art toindicate a nucleic acid or polypeptide sequence possessing a high degreeof sequence relatedness to a subject sequence. Such relatedness may bequantified by determining the degree of identity and/or similaritybetween the sequences being compared. Falling within this generic termare the terms “ortholog” meaning a nucleic acid or polypeptide that isthe functional equivalent of a nucleic acid or polypeptide in anotherspecies, and “paralog” meaning a functionally similar sequence whenconsidered within the same species. Paralogs present in the same speciesor orthologs of the AtACR2 gene in other plant species can readily beidentified without undue experimentation, by molecular biologicaltechniques well known in the art. As used herein, AtSAP10-14 andOsSAP16-17 refer to AtSAP10-14 and OsSAP16-17, respectively, as well astheir homologs and orthologs.

As used herein, “percent homology” of two amino acid sequences or of twonucleic acid sequences is determined using the algorithm of Karlin andAltschul (1990) Proc. Natl. Acad. Sci., U.S.A. 87: 2264-2268. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST nucleotidesearches are performed with the NBLAST program, score=100, word length12, to obtain nucleotide sequences homologous to a nucleic acid moleculeof the invention. BLAST protein searches are performed with the XBLASTprogram, score=50, word length=3, to obtain amino acid sequenceshomologous to a reference polypeptide. To obtain gapped alignments forcomparison purposes, Gapped BLAST is utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters are typically used. (Seehttp://www.ncbi.nlm.nih.gov)

In addition, nucleic acids that are substantially identical to a nucleicacid encoding an AtSAP10-14 or an OsSAP16-17 polypeptide are included.By “substantially identical” is meant a polypeptide or nucleic acidhaving a sequence that is at least about 75%, specifically about 85%,more specifically about 90%, and even more specifically about 95% ormore identical to the sequence of the reference amino acid or nucleicacid sequence. For polypeptides, the length of the reference polypeptidesequence will generally be at least about 16 amino acids, orspecifically at least about 20 amino acids, more specifically at leastabout 25 amino acids, and most specifically at least about 35 aminoacids. For nucleic acids, the length of the reference nucleic acidsequence will generally be at least about 50 nucleotides, specificallyat least about 60 nucleotides, more specifically at least about 75nucleotides, and most specifically at least about 110 nucleotides.

Typically, homologous sequences can be confirmed by hybridization,wherein hybridization under stringent conditions. Using the stringenthybridization (i.e., washing the nucleic acid fragments twice where eachwash is at room temperature for 30 minutes with 2× sodium chloride andsodium citrate (SCC buffer; 150 mM sodium chloride and 15 mM sodiumcitrate, pH 7.0) and 0.1% sodium dodecyl sulfate (SDS); followed bywashing one time at 50° C. for 30 minutes with 2× SCC and 0.1% SDS; andthen washing two times where each wash is at room temperature for 10minutes with 2×SCC), homologous sequences can be identified comprisingat most about 25 to about 30% base pair mismatches, or about 15 to about25% base pair mismatches, or about 5 to about 15% base pair mismatches.

Nucleic acids encoding AtSAP10-14 or OsSAP16-17 sequences allow for thepreparation of relatively short DNA (or RNA) sequences having theability to specifically hybridize to such gene sequences. The shortnucleic acid sequences may be used as probes for detecting the presenceof complementary sequences in a given sample, or may be used as primersto detect, amplify or mutate a defined segment of the DNA sequencesencoding an AtSAP10-14 or OsSAP16-17 polypeptide. A nucleic acidsequence employed for hybridization studies may be greater than or equalto about 14 nucleotides in length to ensure that the fragment is ofsufficient length to form a stable and selective duplex molecule. Suchfragments are prepared, for example, by directly synthesizing thefragment by chemical means, by application of nucleic acid reproductiontechnology, such as PCR technology, or by excising selected nucleic acidfragments from recombinant plasmids containing appropriate inserts andsuitable restriction sites.

The term plant SAP includes nucleic acids that encode the AtSAP10-14 andOsSAP16-17 polypeptides or full-length proteins that containsubstitutions, insertions, or deletions into the polypeptide backbone.Related polypeptides are aligned with AtSAP10-14 and OsSAP16-17 byassigning degrees of homology to various deletions, substitutions andother modifications. Homology can be determined along the entirepolypeptide or nucleic acid, or along subsets of contiguous residues.The percent identity is the percentage of amino acids or nucleotidesthat are identical when the two sequences are compared. The percentsimilarity is the percentage of amino acids or nucleotides that arechemically similar when the two sequences are compared. AtSAP10-14 orOsSAP16-17, and homologous polypeptides are preferably greater than orequal to about 75%, preferably greater than or equal to about 80%, morepreferably greater than or equal to about 90% or most preferably greaterthan or equal to about 95% identical.

A homologous polypeptide may be produced, for example, by conventionalsite-directed mutagenesis of nucleic acids (which is one avenue forroutinely identifying residues of the molecule that are functionallyimportant or not), by random mutation, by chemical synthesis, or bychemical or enzymatic cleavage of the polypeptides.

In the case of polypeptide sequences that are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide that is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

Reference herein to either the nucleotide or amino acid sequence ofAtSAP10-14 and OsSAP16-17 also includes reference to naturally occurringvariants of these sequences. Non-naturally occurring variants thatdiffer from SEQ ID NOs:1-7 (nucleotide) and 8-14 (amino acid) and retainbiological function are also included herein. Preferably the variantscomprise those polypeptides having conservative amino acid changes,i.e., changes of similarly charged or uncharged amino acids. Geneticallyencoded amino acids are generally divided into four families: (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3)non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. As each member of a family has similar physical andchemical properties as the other members of the same family, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the bindingproperties of the resulting molecule. Whether an amino acid changeresults in a functional polypeptide can readily be determined byassaying the properties of transgenic plants containing the AtSAP10-14and OsSAP16-17 derivatives.

Reference to AtSAP10-14 and OsSAP16-17 also refers to polypeptidederivatives of AtSAP10-14 and OsSAP16-17. As used herein, “polypeptidederivatives” include those polypeptides differing in length from anaturally-occurring AtSAP10-14 and OsSAP16-17 and comprising about fiveor more amino acids in the same primary order as is found in AtSAP10-14and OsSAP16-17. Polypeptides having substantially the same amino acidsequence as AtSAP10-14 and OsSAP16-17 but possessing minor amino acidsubstitutions that do not substantially affect the ability of AtSAP10-14and OsSAP16-17 polypeptide derivatives to interact with AtSAP10-14 andOsSAP16-17-specific molecules, respectively, such as antibodies, arewithin the definition of AtSAP10-14 and OsSAP16-17 polypeptidederivatives. Polypeptide derivatives also include glycosylated forms,aggregative conjugates with other molecules and covalent conjugates withunrelated chemical moieties.

In one embodiment, the SAP (e.g., AtSAP10-14 and OsSAP16-17 genes ortheir homologs) are expressed in vectors suitable for in vivo expressionsuch as, for example, plant expression systems. The SAP nucleic acidsare inserted into a recombinant expression vector or vectors. The term“recombinant expression vector” refers to a plasmid, virus, or othermeans known in the art that has been manipulated by insertion orincorporation of the SAP genetic sequence. The term “plasmids” generallyis designated herein by a lower case p preceded and/or followed bycapital letters and/or numbers, in accordance with standard namingconventions that are familiar to those of skill in the art. Plasmidsdisclosed herein are either commercially available, publicly availableon an unrestricted basis, or can be constructed from available plasmidsby routine application of well-known, published procedures. Manyplasmids and other cloning and expression vectors are well known andreadily available, or those of ordinary skill in the art may readilyconstruct any number of other plasmids suitable for use. These vectorsare transformed into a suitable host cell to form a host cell vectorsystem for the production of a polypeptide.

The term recombinant nucleic acid or nucleic acid refers to a nucleicacid that is made by the combination of two otherwise separated segmentsof sequence accomplished by the artificial manipulation of isolatedsegments of nucleic acids by genetic engineering techniques or bychemical synthesis. In so doing, one may join together nucleic acidsegments of desired functions to generate a desired combination offunctions.

The term transgene refers to an isolated nucleic acid or nucleic acidthat comprises a coding sequence encoding a protein or RNA molecule.

The SAP nucleic acids are inserted into a vector adapted for expressionin a plant, bacterial, yeast, insect, amphibian, or mammalian cell thatfurther comprises the regulatory elements necessary for expression ofthe nucleic acid molecule in the plant, bacterial, yeast, insect,amphibian, or mammalian cell operatively linked to the nucleic acidmolecule encoding SAP. Suitable vectors for plant expression includeT-DNA vectors. “Operatively linked” refers to a juxtaposition whereinthe components so described are in a relationship permitting them tofunction in their intended manner. An expression control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with theexpression control sequences. As used herein, the term “expressioncontrol sequences” refers to nucleic acid sequences that regulate theexpression of a nucleic acid sequence to which it is operatively linked.Expression control sequences are operatively linked to a nucleic acidsequence when the expression control sequences control and regulate thetranscription and, as appropriate, translation of the nucleic acidsequence. Thus, expression control sequences can include appropriatepromoters, enhancers, transcription terminators, a start codon (i.e.,ATG) in front of a protein-encoding gene, splicing signals for introns(if introns are present), maintenance of the correct reading frame ofthat gene to permit proper translation of the mRNA, and stop codons. Theterm “control sequences” is intended to include, at a minimum,components whose presence can influence expression, and can also includeadditional components whose presence is advantageous, for example,leader sequences and fusion partner sequences. Expression controlsequences can include a promoter. By “promoter” is meant minimalsequence sufficient to direct transcription. Also included are thosepromoter elements which are sufficient to render promoter-dependent geneexpression controllable for cell-type specific, tissue-specific, orinducible by external signals or agents; such elements may be located inthe 5′ or 3′ regions of the gene. Both constitutive and induciblepromoters are included. If a promoter is inducible, there are sequencespresent that mediate regulation of expression so that the associatedsequence is transcribed only when an inducer (e.g., light) is availableto the plant or plant tissue. An exemplary promoter is the ArabidopsisACT2 promoter that is constitutively active and provides high levels ofexpression of an associated coding sequence.

Other suitable expression control sequences include 3′ untranslatedsequences located downstream of an associated coding sequence. Anexemplary 3′ untranslated sequence is that from the ACT2 gene ofArabidopsis.

With respect to a coding sequence, the term “plant-expressible” meansthat the coding sequence (nucleotide sequence) can be efficientlyexpressed by plant cells, tissue and/or whole plants. As used herein, aplant-expressible coding sequence has a GC composition consistent withacceptable gene expression in plant cells, a sufficiently low CpGcontent so that expression of that coding sequence is not restricted byplant cells, and codon usage that is consistent with that of plantgenes. Where it is desired that the properties of the plant-expressiblemetal resistance gene are identical to those of the naturally occurringmetal resistance gene, the plant-expressible homolog will have asynonymous coding sequence or a substantially synonymous codingsequence. A substantially synonymous coding sequence is one in thatthere are codons that encode similar amino acids to a comparisonsequence, or if the amino acid substituted is not similar in propertiesto the one it replaces, that change has no significant effect onenzymatic activity for at least one substrate of that enzyme. Asdiscussed herein, it is well understood that in most cases, there issome flexibility in amino acid sequence such that function is notsignificantly changed. Conservative changes in amino acid sequence, andthe resultant similar protein can be readily tested using proceduressuch as those disclosed herein. Where it is desired that theplant-expressible gene have different properties, there can be variationin the amino acid sequence as compared to the wild type gene, and theproperties of metal resistance can be readily determined as describedherein.

“Plant-expressible transcriptional and translational regulatorysequences” are those that can function in plants, plant tissue and/orplant cells to effect the transcriptional and translational expressionof the nucleotide sequences with that they are associated. Included are5′ sequences that qualitatively control gene expression (turn on or offgene expression in response to environmental signals such as light, orin a tissue-specific manner) and quantitative regulatory sequences thatadvantageously increase the level of downstream gene expression. Anexample of a sequence motif that serves as a translational controlsequence is that of the ribosome binding site sequence. Polyadenylationsignals are examples of transcription regulatory sequences positioneddownstream of a target sequence. Exemplary flanking sequences includethe 3′ flanking sequences of the nos gene of the Agrobacteriumtumefaciens Ti plasmid.

The plant-expressible transcription regulatory sequence optionallycomprises a constitutive promoter to drive gene expression throughoutthe whole plant or a majority of plant tissues. In one embodiment, theconstitutive promoter drives gene expression at a higher level than theendogenous plant gene promoter. In one embodiment, the constitutivepromoter drives gene expression at a level that is at least two-foldhigher, specifically at least five-fold higher, and more specifically atleast ten-fold higher than the endogenous plant gene promoter. Suitableconstitutive promoters include plant virus promoters such as thecauliflower mosaic virus (CaMV) 35S and 19S promoters. An exemplaryplant virus promoter is the cauliflower mosaic virus 35S promoter.Suitable constitutive promoters further include promoters for plantgenes that are constitutively expressed such as the plant ubiquitin,Rubisco, and actin promoters such as the ACT 1 and ACT2 plant actingenes. Exemplary plant gene promoters include the ACT2 promoter fromArabidopsis (SEQ ID. NO:15) and the ACT1 promoter from rice (GenBankAccession no. 544221.1; SEQ ID. NO:16).

Where a regulatory element is to be coupled to a constitutive promoter,generally a truncated (or minimal) promoter is used, for example, thetruncated 35S promoter of Cauliflower Mosaic Virus. Truncated versionsof other constitutive promoters can also be used to provide CAAT andTATA-homologous regions; such promoter sequences can be derived fromthose of Agrobacterium tumefaciens T-DNA genes such as nos, ocs and masand plant virus genes such as the CaMV 19S gene or the ACT2 gene ofArabidopsis. Translational control sequences specifically exemplifiedherein are the nucleotides between 8 and 13 upstream of the ATGtranslation start codon for bacterial signals and from nucleotides 1 to7 upstream of the ATG translation start codon for plants.

A minimal promoter contains the DNA sequence signals necessary for RNApolymerase binding and initiation of transcription. For RNA polymeraseII promoters, the promoter is identified by a TATA-homologous sequencesmotif about 20 to 50 base pairs upstream of the transcription start siteand a CAAT-homologous sequence motif about 50 to 120 base pairs upstreamof the transcription start site. By convention, the nucleotides upstreamof the transcription start with increasingly large numbers extendingupstream of (in the 5′ direction) from the start site. In oneembodiment, transcription directed by a minimal promoter is low and doesnot respond either positively or negatively to environmental ordevelopmental signals in plant tissue. An exemplary minimal promotersuitable for use in plants is the truncated CaMV 35S promoter, thatcontains the regions from −90 to +8 of the 35S gene. Where high levelsof gene expression are desired, transcription regulatory sequences thatupregulate the levels of gene expression may be operatively linked to aminimal promoter is used thereto. Such quantitative regulatory sequencesare exemplified by transcription enhancing regulatory sequences such asenhancers.

In one embodiment, the plant-expressible transcription regulatorysequence comprises a tissue or organ-specific promoter to drive geneexpression in selected organs such as roots or shoots and tissuestherein. In one embodiment, the organ-specific promoter drives geneexpression in below ground tissues such as roots and root hairs. In oneembodiment, the organ-specific promoter drives gene expression in aboveground tissues such as shoots and leaves. An exemplary leaf-specificpromoter is the SRS1 promoter (SEQ ID. NO:17). In one embodiment, theorgan-specific promoter drives gene expression in floral andreproductive tissues.

The plant-expressible transcription regulatory sequence optionallycomprises an inducible promoter to drive gene expression in response toselected stimuli. Suitable inducible promoters include a light induciblepromoter such as the SRS1 promoter, arsenic inducible promoters such asthe OsACR2 promoter, and the chlorophyll A/B binding proteinlight-inducible transcription regulatory sequences.

The choice of vector used for constructing the recombinant DNA moleculedepends on the functional properties desired, e.g., replication, proteinexpression, and the host cell to be transformed. In one embodiment, thevector comprises a prokaryotic replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extrachromosomally when introduced into aprokaryotic host cell, such as a bacterial host cell. In addition, thevector may also comprise a gene whose expression confers a selectiveadvantage, such as a drug resistance, to the bacterial host cell whenintroduced into those transformed cells. Suitable bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline, among other selective agents. The neomycinphosphotransferase gene has the advantage that it is expressed ineukaryotic as well as prokaryotic cells.

Vectors typically include convenient restriction sites for insertion ofa recombinant DNA molecule. Suitable vector plasmids include pUC8, pUC9,pBR322, and pBR329 available from BioRad Laboratories (Richmond, Calif.)and pPL, pK and K223 available from Pharmacia (Piscataway, N.J.), andpBLUESCRIPT® and pBS available from Stratagene (La Jolla, Calif.).Suitable vectors include, for example, Lambda phage vectors includingthe Lambda ZAP vectors available from Stratagene (La Jolla, Calif.).Other exemplary vectors include pCMU. Other appropriate vectors may alsobe synthesized, according to known methods; for example, vectorspCMU/K^(b) and pCMUII which are modifications of pCMUIV.

Suitable expression vectors capable of expressing an isolated nucleicacid sequence in plant cells and capable of directing stable integrationwithin the host plant cell include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens, and severalother expression vector systems known to function in plants. See forexample, Verma et al., No. WO87/00551, incorporated herein by reference.

Expression and cloning vectors optionally contain a selectable marker,that is, a gene encoding a protein necessary for the survival or growthof a host cell transformed with the vector. Although such a marker genemay be carried on another nucleic acid sequence co-introduced into thehost cell, it is most often contained on the cloning vector. Only thosehost cells into which the marker gene has been introduced will surviveand/or grow under selective conditions. Suitable selection genes encodeproteins that (a) confer resistance to antibiotics or other toxicsubstances, e.g., ampicillin, neomycin, methotrexate, etc.; (b)complement auxotrophic deficiencies; or (c) supply critical nutrientsnot available from complex media. The choice of the proper selectablemarker will depend, in part, on the host cell. In one embodiment, themetal resistance coding sequence itself is used as a selectable markerto select transformants on medium supplemented with an appropriateconcentration of arsenic.

In one embodiment, the plant SAP coding sequence is cloned into a vectorsuitable for expression in Arabidopsis and rice under the control ofdifferent constitutive promoters including the CaMV 35S promoter and theactin promoters from Arabidopsis and rice. In one embodiment, the plantSAP coding sequence is regulated by an organ or tissue-specific or aninducible promoter. An exemplary tissue-specific promoter is theleaf-specific SRS1 promoter (SEQ ID. NO:14). In one embodiment, theplant SAP coding sequence is cloned into a plant expression cassetteconstruct or vector comprising a promoter, convenient cloning sites andthe nos transcription terminator (NOSt).

Transformation of a host cell with an expression vector or other DNA iscarried out by conventional techniques as are well known to thoseskilled in the art. By “transformation” is meant a permanent ortransient genetic change induced in a cell following incorporation ofnew DNA (i.e., DNA exogenous to the cell). Where the cell is a plantcell, a permanent genetic change is generally achieved by introductionof the DNA into the genome of the cell. By “transformed cell” or “hostcell” is meant a cell (e.g., prokaryotic or eukaryotic) into which (orinto an ancestor of which) has been introduced, by means of recombinantDNA techniques, a DNA molecule encoding an SAP (e.g., an AtSAP10-14 orOsSAP16-17 polypeptide), or fragment thereof.

Recombinant host cells, in the present context, are those that have beengenetically modified to contain an isolated DNA molecule. The DNA can beintroduced by a means that is appropriate for the particular type ofcell, including without limitation, transfection, transformation,lipofection, or electroporation.

Also included herein are transgenic plants that have been transformedwith an SAP gene. A “transgenic plant” is one that has been geneticallymodified to contain and express recombinant DNA sequences, either asregulatory RNA molecules or as proteins. As specifically exemplifiedherein, a transgenic plant is genetically modified to contain andexpress a recombinant DNA sequence operatively linked to and under theregulatory control of transcriptional control sequences that function inplant cells or tissue or in whole plants. As used herein, a transgenicplant also encompasses progeny of the initial transgenic plant wherethose progeny contain and are capable of expressing the recombinantcoding sequence under the regulatory control of the plant-expressibletranscription control sequences described herein. Seeds containingtransgenic embryos are encompassed within this definition.

Individual plants within a population of transgenic plants that expressa recombinant gene may have different levels of gene expression. Thevariable gene expression is due to multiple factors including multiplecopies of the recombinant gene, chromatin effects, and gene suppression.Accordingly, a phenotype of the transgenic plant may be measured as apercentage of individual plants within a population. In one embodiment,greater than or equal to about 25% of the transgenic plants express thephenotype. Specifically, greater than or equal to about 50% of thetransgenic plants express the phenotype. More specifically, greater thanor equal to about 75% of the transgenic plants express the phenotype. Inone embodiment, the phenotype is metal resistance. In anotherembodiment, the phenotype is metal resistance and stress resistance.

The transgenic plant is transformed with an isolated nucleic acid ornucleic acid molecule comprising a plant SAP coding sequence operativelylinked to a plant-expressible transcription regulatory sequence.Exemplary plant SAP genes include Arabidopsis AtSAP10-14 (SEQ IDNOs:1-5) and rice OsSAP16-17 (SEQ ID NOs:6-7). The transgenic plantexpresses a plant SAP. Suitable plant SAPs include SAPs fromArabidopsis, rice, and Brassica plants. Exemplary plant SAPs includeArabidopsis AtSAP10-14 (SEQ ID NOs:8-12) and rice OsSAP16-17 (SEQ IDNOs:13-14).

The present inventors have transformed plants with recombinant DNAmolecules that encode a plant SAP. Transgenic plants and plant cellsexpressing the recombinant plant SAP gene are more resistant to metalsthan wild type control plants. In one embodiment, greater than or equalto about 25% of the transgenic plants are resistant to a concentrationof metal that is lethal to wild type control plants. Specifically,greater than or equal to about 50%, and more specifically, greater thanor equal to about 75%, of the transgenic plants are resistant to aconcentration of metal that inhibits growth in wild type control plants.

Transgenic plants and plant cells expressing the recombinant plant SAPgene are more resistant to environmental stresses than wild type controlplants. In one embodiment, greater than or equal to about 25% of thetransgenic plants are resistant to an environmental stress that inhibitsgrowth in wild type control plants. Specifically, greater than or equalto about 50%, and more specifically, greater than or equal to about 75%,of the transgenic plants are resistant to an environmental stress thatinhibits growth in wild type control plants.

The increase in metal resistance in the transgenic plants also leads toincreased biomass when the transgenic plants are grown in the presenceof a concentration of metal that inhibits growth in wild type controlplants. The term “biomass” refers to the biological material in plantsand includes internal plant structures that comprise dead cells, such asxylem. In one embodiment, biomass is measured by the dry weight of aplant. In one embodiment, the total biomass of the transgenic plant isgreater than or equal to about 100%; specifically, greater than or equalto about 250%; and more specifically, greater than or equal to about500% of the total biomass of wild type control plants when grown in thepresence of a concentration of metal that inhibits growth in wild typecontrol plants.

A recombinant DNA construct including a plant-expressible gene or otherDNA of interest is inserted into the genome of a plant by a suitablemethod. Suitable methods include, for example, Agrobacteriumtumefaciens-mediated DNA transfer, direct DNA transfer,liposome-mediated DNA transfer, electroporation, co-cultivation,diffusion, particle bombardment, microinjection, gene gun, calciumphosphate coprecipitation, viral vectors, and other techniques. Suitableplant transformation vectors include those derived from a Ti plasmid ofAgrobacterium tumefaciens. In addition to plant transformation vectorsderived from the Ti or root-inducing (Ri) plasmids of Agrobacterium,alternative methods can be used to insert DNA constructs into plantcells. A transgenic plant can be produced by selection of transformedseeds or by selection of transformed plant cells and subsequentregeneration.

Techniques are well known to the art for the introduction of DNA intomonocots as well as dicots, as are the techniques for culturing suchplant tissues and regenerating those tissues. Monocots that have beensuccessfully transformed and regenerated include wheat, corn, rye, riceand asparagus. For efficient regeneration of transgenic plants, it isdesired that the plant tissue used in the transformation possess a highcapacity to produce shoots. For example, Aspen stem sections have goodregeneration capacity. Poplars have been successfully transformed andregenerated as have cottonwoods.

In one embodiment, a recombinant DNA, such as a transgene construct, isintroduced into rice plants. Transformed rice cells are selected andregenerated into transgenic rice plants. In one embodiment, transformedrice cells are selected on media containing an appropriate antibiotic.The rice cells are induced to form a somatic embryogenic callus. Thecallus is treated with the appropriate reagents such as plant hormonesto induce the formation of root and shoot tissue. In this manner,transgenic rice plants can be regenerated from the callus derived fromtransformed rice cells.

In one embodiment, the plant SAP coding sequence is subcloned under thecontrol of the soybean plant ribulose biphosphate carboxylase (Rubisco)small subunit promoter SRS1 and the 3′ nos terminator in pBluescript®.This coding sequence and promoter are previously shown to be stronglytranscriptionally induced in leaves by light. Expression directed bythis promoter is very low in roots. The entire chimeric gene includingthe SRS1 promoter, the SAP coding sequence, and the 3′ nos transcriptionterminator sequence, is subcloned into the plant expression T-DNA binaryvector pBIN19, that has the selectable kanamycin-resistance marker(NPTII). A. thaliana is transformed using vacuum infiltrationtechnology, and the T1 generation seeds are screened for kanamycinresistance. Transgenic plants transformed with an isolated SAP nucleicacid are produced. In one embodiment, the plant also expresses aphytochelatin biosynthetic enzyme coding sequence, e.g., γ-ECS, PSand/or GS.

The transgenic plant optionally further comprises an isolated nucleicacid suitable for expression of a phytochelatin biosynthetic enzymecoding sequence. In another embodiment, the arsenic-resistant transgenicplants also overexpress thiol-rich peptides like glutathione andphytochelatins to further improve arsenic tolerance. Phytochelatins(PCs) are small peptides that are synthesized non-ribosomally fromcommon amino acid precursors in a three-step enzymatic pathway. Suitablegenes that encode phytochelatins include the prokaryoticgamma-glutamylcysteine synthase (γECS) and glutathione synthase (GS)genes and the eukaryotic phytochelatin synthase (PCS) genes. Exemplaryphytochelatin genes include the E. coli γECS (GenBank Accession no.X03954; SEQ ID NO. 18) and GS (GenBank Accession no. 28377; SEQ ID NO.19) genes and the PCS genes from fission yeast (Schizosaccharomycespombe) (GenBank Accession no. 28377; SEQ ID NO. 20). In one embodiment,the phytochelatin biosynthetic enzyme coding sequence is greater than orequal to about 75%, 85%, 90% or 95% homologous with a sequence selectedfrom the group consisting of SEQ ID NO:18, SEQ ID NO:19, and SEQ IDNO:20, wherein the phytochelatin biosynthetic enzyme coding sequence hasphytochelatin biosynthesis activity. Plants that co-express aphytochelatin synthetic gene such as γECS, GS and PCS together with anSAP gene are further improved in metal resistance. In one embodiment,phytochelatin biosynthetic genes are overexpressed in roots. Withoutbeing bound by theory, it is believed that by overexpressingphytochelatin biosynthetic genes in roots, the thiol-rich peptides willbind arsenite generated in roots and thus improve arsenic tolerance andfurther prevent the movement of arsenic to the aboveground tissues.

In one embodiment, the levels of PC pathway intermediates (gamma-EC, GSHand PC) are expressed at a level in excess of 1% of the total cellprotein. In this example, three vector systems are used for all three PCsynthesizing enzymes in order to compare their activity and to avoidpotential co-suppression problems. For strong constitutive expressionand as an alternative promoter to the CaMV 35S promoter, a novel actinpromoter expression vector, ACT2pt was developed. The ACT2pt comprisesthe promoter (p) and terminator (t) from the constitutive ACT2 gene. Incontrolled experiments with 30 independent ACT2pt/reporter lines and 30independent 35Sp/reporter lines, the ACT2pt vector gives about 5-10times higher levels of reporter expression than the 35Sp vector. Inseveral independent experiments using the ACT2pt vector, co-suppressionof the endogenous ACT2 gene or the transgene was not observed, even whenmultiple copies are present. While a plant with low levels of ACT2ptdriven expression was not obtained, approximately 10-20% of the 35Spplants had no detectable reporter expression. Furthermore, the lowestACT2pt plants are equivalent to the highest 35Sp plants. This apparentinsensitivity to cosuppression offers a significant advantage in themultigene strategy being used.

In one embodiment, the nucleic acids encoding thiol-rich peptides aremodified by PCR to comprise appropriate sites for cloning to makein-frame translational fusions with actin and SRS1 light regulatedpromoters. In one embodiment, the nucleic acids are modified fordetection in E. coli and plants. Monoclonal antibodies specific toAtACR2, γECS, GS, and PCS (fission yeast) proteins have been generatedto monitor protein expression. The Arabidopsis PCS protein was taggedwith an HA (hemagglutinin) epitope to allow monitoring with acommercially available HA-specific antibody. All four proteins conferincreased metal tolerance to E. coli, when expressed under the controlof the lac promoter in pBluescript® vectors. In one embodiment, all fourgenes are derived from plants including, for example, Arabidopsis andrice. Without being bound by theory, it is believed that thiol-richpeptides such as glutathione and phytochelatins, bind arsenic andcontribute to arsenic tolerance and accumulation. It is believed thatthe GS-As and PC-As complexes are pumped into vacuoles for storage, thusimproving arsenic tolerance.

In one embodiment, the transgenic plants overexpress a plant SAP and athiol-rich peptide to synergistically improve metal resistance. Theoverexpression of the plant SAP improves the As(III) binding capacity ofthe plant cells while the overexpression of the thiol-rich peptidesprovide thiol sinks for As(III). For example, the transgenic plantco-overexpresses heterologous PC synthetic genes and plant SAP. In oneembodiment, the transgenic plant overexpresses the heterologous PCsynthetic genes and plant SAP in a tissue-specific manner. Suitabletissues include, for example, roots, leaves, shoots, stems, and seeds.

In one embodiment, transgenic plants are transformed with vectors thatprovide overexpression of thiol-rich peptides. For example, the ACT2ptvector has been used to drive exceptionally high levels of constitutivetransgenic expression of GS throughout the plant. The ACT2pt vector mayfurther contain intron (IVSL) that enhances expression 20-fold. The ACT2poly(A) region (Act2t) ensures efficient transcription termination, andit contains multiple polyadenylation sites.

The following examples are provided for illustrative proposes and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified compositions and methods, that occur tothe skilled artisan, are intended to fall within the scope of thepresent invention.

EXAMPLES Plasmids

The pBluescript® SK (−) plasmid (Stratagene, La Jolla, Calif.) and twoT-DNA vectors, promoterless binary vectors pBIN19 (Clontech, Palo Alto,Calif.) and pCambia (Cambia, Canberra, Australia), that are designed forexpressing genes under a promoter of interest by Agrobacterium-mediatedtransformations, are obtained commercially.

Example 1 Cloning SAP Genes

Various plant databases are searched for genes encoding amino acidsequence homologs of the previously described C. elegans aip-1 SAP.Sequences that are the likely plant homologs of the aip-1 proteinsequence are identified. Five homologous genes are identified in A.thaliana (Columbia ecotype) and two genes are identified in rice (Oryzasativa (japonica cultivar)). Three of the putative As(III)-bindingproteins from Arabidopsis, AtSAP11 (SEQ ID NO:8), AtSAP13 (SEQ ID NO:9),AtSAP12 (SEQ ID NO:11), and the two putative As(III)-binding proteinsfrom rice, OsSAP16 (SEQ ID NO:13) and OsSAP17 (SEQ ID NO:14) have twoconserved AN1 zinc finger domains and a C₂H₂ domain. High sequencehomology among these amino acid sequences and specific arrangements ofconserved Cys and His residues suggest that their functions are wellconserved. These Arabidopsis and rice SAP protein sequences are highlycysteine and histidine-rich and have more than 60% similarity to theanimal protein in the conserved sulfur-rich region thought to bindAs(III). Alignment of the predicted protein sequences of all AtSAPs andrice OsSAP16 revealed the presence of highly conserved cysteine andhistidine repeats arranged in particular configurations. For example,Arabidopsis AtSAP11 sequences contain a distinct pattern of 19 conservedcysteine residues in a 279 amino acids protein and 18, 9, 16, and 12conserved cysteine residues in AtSAP13, AtSAP14, AtSAP12, and AtSAP10proteins, respectively. Rice OsSAP16 contains 21 cysteine and 10histidine conserved residues. These Cys- and His-residues are arrangedin specific orders that form typical metal-binding domains. Theseproteins have several CX₂C, CX₄C, CX₂CX₄C and CXHX₅HXC domains, whichmay bind As(III) and other heavy metals, and thus provide resistance.The phylogenetic analysis of several SAP proteins show that ArabidopsisAtSAP11, AtSAP13, and rice OsSAP16 are closely related to each other,whereas, AtSAP14 and AtSAP12 are separated from this group and isdistantly related. The AtSAP11, AtSAP13 and OsSAP16 have extendedC-terminal where they contain extra C₂H₂ domain. The C₂H₂ domain ismissing in AtSAP14 and AtSAP12 protein sequences. AtSAP14 and AtSAP12are tested in order to determine if SAPs that lack the C-terminal C₂H₂type zinc finger domain have different functions than other SAPs.

The Arabidopsis SAP sequences are cloned into the multiple cloning siteregion of pBluescript® II SK (Stratagene) to make a bacterial expressionplasmid pBS/AtSAP. For plant expression, the plant SAP sequences aresubcloned under the regulatory control of the Arabidopsis actin ACT2promoter and the nopaline synthase (nos) 3′ terminator to createpACT2p/SAP. The entire cassette containing the promoter, AtACR2 codingsequence and nos 3′ terminator, is subcloned into the AgrobacteriumpBIN19 Ti vector for transformation into plants.

The Arabidopsis AtSAP13 gene is amplified and cloned from Arabidopsisshoot and root cDNA libraries. The Arabidopsis shoot and root cDNAlibraries are made after 12 and 24 hrs induction with 150 micromolarsodium arsenate and 40 micromolar sodium arsenite. AtSAP13 is PCRamplified using sense primer,5′-TACGTCGGATCCTAAGGAGGATAGACCATGGGAACTCCAGAATTTCCA GATCTGGGTA-3′ (SEQID NO:23) and the antisense primer,5′-TAGCTGGAGCTCAAGCTTCTCGAGCTAGGCTTTAGAAGTGCCACGATGAT CCTTAT-3′ (SEQ IDNO:24). The Arabidopsis AtSAP11, AtSAP14, AtSAP12, and AtSAP10 genes areamplified and cloned using similar strategies. The PCR is carried out as1 cycle at 94° C. for 2 min followed by 40 cycles with denaturing,annealing and extending temperatures and times of 94° C. for 1 min, 55°C. for 1 min and 72° C. for 1 min with an additional extension cycle of72° C. for 10 min. The amplified fragment corresponding to each gene isgel extracted and then cloned into TOPO TA cloning vector (Invitrogen).The isolated plasmid is sequenced and nucleotide sequences are analyzedand confirmed using Sequencher (GeneCode Corporation)

AtSAP11 forward (sense) primer (SEQ ID NO: 21)5′-TACGTCGAATTCAGGAGGTAGACCATGGGGACTCCGGAATT-3′;reverse (anti-sense) primer (SEQ ID NO: 22)5′-TAGCTGGTCGACAAGCTTCTATGCTTTCGAAGTGCCT-3′.AtSAP14 forward (sense) primer (SEQ ID NO: 25)5′-TACGTCGAATTCAGGAGGTAGACCATGGGGACTCCGGAATT-3′;reverse (anti-sense) primer (SEQ ID NO: 26)5′-TAGCTGGTCGACAAGCTTTTATTCTTCTTCCCATTCAACAT-3′.AtSAP12 forward (sense) primer (SEQ ID NO: 27)5′-TACGTCGGATCCAGGAGGTAGACCATGGCAGGAGGAGGAACAGAAG CGT-3′;reverse (anti-sense) primer (SEQ ID NO: 28)5′-TAGCTGGAATTCCTAAAACGATCTAACTGATGGT-3′. AtSAP10 forward (sense) primer(SEQ ID NO: 29) 5′-TACGTCGGATCCAGGAGGTAGACCATGGTGAACGAAACAGAAGCA T-3′;reverse (anti-sense) primer (SEQ ID NO: 30)5′-TAGCTGCTCGAGAAGCTTCTAAAACCTCTGCAACTTGTCA-3′.

For overexpression of AtSAP13 in plants, the NcoI-XhoI fragment of thisgene is cloned under a strong constitutive expression vector cassette,pACT2pt. The expression vector pACT2pt has Arabidopsis ACT2 genepromoter and ACT2 gene terminator. The KpnI-SacI fragment containing theentire gene cassette (ACT2pt/AtSAP) is taken out and subcloned intopBIN19 binary vector for transformation into Agrobacterium strain C58.The Arabidopsis plants are transformed with this construct via standardflower dip using vacuum infiltration. The kanamycin resistant transgenicplants are selected on MS media supplemented with kanamycin. The cloningstrategy for AtSAP13 is depicted schematically in FIG. 2. The otherAtSAP genes are subcloned into suitable vector cassettes using similarstrategies.

The rice OsSAP16 gene is amplified and cloned from rice cDNA libraries.The rice shoot and root cDNA libraries are made after 24 h inductionwith 300 micromolar sodium arsenate and 100 micromolar sodium arsenite.The 290 amino acid OsSAP16 gene is PCR amplified using sense primer,5′-TACGTCGGATCCGGACTAAAGGAGGCCATGGGGACGCCGGAGTTCCCCA-3′ (SEQ ID NO:31)and the antisense primer,5′-TAGCTGCTCGAGCTACGCTCTTGACGTTCCTCCGTGGTCCCTCT-3′ (SEQ ID NO:32). ThePCR is carried out as 1 cycle at 94° C. for 2 min followed by 40 cycleswith denaturing, annealing and extending temperatures and times of 94°C. for 1 min, 55° C. for 1 min and 72° C. for 2 min with an additionalextension cycle of 72° C. for 10 min. The PCR amplified OsSAP16 gene iscloned in pBluescriptII SK using BamHI-XhoI combination of restrictionenzymes. For over expression of OsSAP in plants, the NcoI-XhoI fragmentof this gene is subcloned into an expression vector pACT1p/NOSt. Theexpression vector pACT1p/NOSt has the rice ACT1 gene promoter and NOSgene terminator. The KpnI-SacI fragment containing the entire genecassette (ACT1p/OsSAP16/NOSt) is isolated and subcloned into the pCambiabinary vector for transformation into Agrobacterium strain LBA4404.OsSAP17 is cloned and amplified using a similar strategy. The OsSAP17forward primer is 5′-TACGTCGGATCCGGACTAAAGGAGGCCATGGCGCGGCGGGGCACGGA-3′(SEQ ID NO:33) and the OsSAP17 forward primer is5′-TAGCTGCTCGAGTCAGAAAATCTTCATGTTT-3′ (SEQ ID NO:34).

Example 2 Cloning γ-ECS, GS, and PS for Bacterial Expression

The γ-ECS (GenBank Accession no. X03954; SEQ ID NO. 18) and GS (GenBankAccession no. 28377; SEQ ID NO. 19) genes are amplified by PCR, usingsynthetic primers, from genomic DNA of E. coli SK1592. The fission yeastSchizosaccharomyces pombe PS gene (GenBank Accession no. Z68144; SEQ IDNO. 20) is amplified from a plasmid PsPC/YES clone provided by JulianSchroeder (University of California, San Diego, Calif.). The twooligonucleotide primers for each gene add synthetic flanking sequencesfor cloning and bacterial expression. The sense primers containrestriction endonuclease cloning sites XhoI and NcoI, a TAA stop codon,and bacterial translation signals. The antisense primers contain cloningsites BamHI and HindIII. The PCR products encoding all three genes arecloned first into the XhoI/BamHI replacement region of pBluescript®KS(II) (Stratagene, La Jolla, Calif.) and electroporated into E. colistrain Top10F (Invitrogen, Carlsbad, Calif.). The fidelity of theamplified coding sequences are confirmed by sequencing. To expresshigher levels of protein, the three genes are subcloned into the NdeI(blunt end)/BamHI replacement region of the expression vector pET15b(Novagen, Madison, Wis.) using post-ligation-digestion with XhoI toselect against the parent pET15b vector. These plasmids are expressed inE. coli strain BL121 (Novagen) as per the manufacturer's instructions.

Example 3 Construction of Transgenic Arabidopsis Plants

Plasmid pBIN/AtSAP13, carrying the chimeric plant SAP gene(ACT2p:AtSAP13:ACT2 3′), is electroporated into cells of the C58Agrobacterium tumefaciens strain (GIBCO/BRL, Gaithersburg, Md.).Transformants are verified by using Southern blotting and/or PCR andcultured in YEP medium (10 g/liter Bacto peptone (Difco, Detroit,Mich.)/10 g/liter yeast extract/5 g/liter NaCl) in the presence ofstreptomycin and kanamycin to maintain the T-DNA and pBIN19 plasmids,respectively. Wild type A. thaliana (ecotype Columbia) plants aretransformed with the recombinant A. tumefaciens strains using the vacuuminfiltration procedure.

Example 4 Construction of Transgenic Japonica Rice Plants

Mature japonica cv. Nipponbare rice seeds are dehusked, surfacesterilized and placed onto callus induction medium. The callus tissuederived from the mature embryos are used as the starting material fortransformation. Agrobacterium tumefaciens strain LBA4404 contained thestandard binary vector pCAMBIA1300 harboring the AtACR2 gene under riceACT1 promoter and nos terminator. The plant selectable marker genehygromycin phosphotransferase (hpt) is driven by the cauliflower mosaicvirus (CaMV) promoter.

Media:

Callus induction medium: 30 g/L sucrose, N6 salts and vitamins, 1 g/Lcasein hydrolysate, 0.5 g/L L-proline, 0.5 g/L glutamine, 2 mg/L 2,4-Dand 4 g/L gelrite (pH 5.8).

-   -   Regeneration medium: 30 g/L sucrose, MS salts and vitamins, 1        g/L casein hydrolysate, 2 mg/L BAP, 0.5 mg/L NAA and 4 g/L        gelrite (pH 5.8).    -   Rooting and shoot multiplication medium: 30 g/L sucrose, MS        salts and vitamins and 4 g/L gelrite (pH 5.8).    -   Infection medium: 68.4 g/L sucrose, 36 g/L glucose, N6 salts and        vitamins, 1 g/L casein hydrolysate, 0.5 g/L L-proline, 0.5 g/L        glutamine, 2 mg/L 2,4-D (pH 5.2). Acetosyringone (AS 100 μM) is        added just prior to use.    -   Co-cultivation medium: 30 g/L sucrose, 10 g/L glucose, N6 salts        and vitamins, 1 g/L casein hydrolysate, 0.5 g/L L-proline, 0.5        g/L glutamine, 2 mg/L 2,4-D, 4 g/L gelrite (pH 5.8).        Acetosyringone (AS 100 μM) is added just prior to use.    -   Selection medium I: 30 g/L sucrose, N6 salts and vitamins, 1 g/L        casein hydrolysate, 0.5 g/L L-proline, 0.5 g/L glutamine, 2 mg/L        2,4-D and 4 g/L gelrite (pH 5.8). 300 mg/L cefotaxime and 50        mg/L hygromycin are added to this medium after autoclaving.    -   Selection medium II: 30 g/L sucrose, MS salts and vitamins, 1        g/L casein hydrolysate, 2 mg/L BAP, 0.5 mg/L NAA and 4 g/L        gelrite (pH 5.8). 200 mg/L cefotaxime and 50 mg/L hygromycin are        added to this medium after autoclaving.

Callus Induction

-   -   Rice seeds are dehusked, pre-rinsed with 70% ethanol for 2        minutes and washed with twice with sterile water. The seeds are        then soaked in 0.1% HgCl₂ in a 125 ml sterile conical flask and        placed on a shaker for 30 minutes. The seeds are washed 5 times        with sterile water, dried on sterile filter paper. The surface        sterilized seeds are then kept on callus induction medium (15        seeds per plate) and incubated in light at 25° C. After 2-3        weeks, developing callus is visible on the scutellum of the        mature seed. Calli are sub-cultured to fresh induction medium        and allowed to proliferate.    -   Agrobacterium infection: A single colony of Agrobacterium        tumefaciens strain LBA4404 containing the gene cassette is grown        in 5 ml YEP medium (5 g/L yeast extract, 10 g/L peptone, 5 g/L        NaCl) containing 50 mg/L rifampicin, 100 mg/L kanamycin and used        as inoculum for 50 ml overnight culture. Overnight grown        Agrobacterium culture is adjusted to OD600 0.5 with infection        medium. The liquid infection medium is supplemented with 100 μM        acetosyringone (AS). The calli are infected with this medium for        1 hour in conical flasks on a shaker (low setting).    -   After infection, the bacterial suspension is removed. The calli        are blotted dry on sterile filter paper and placed on        co-cultivation medium. The calli are co-cultivated in dark at        25° C. for 3 days.    -   The infected calli are washed 5 times with sterile water,        blotted dry on sterile filter paper and transferred to selection        medium containing 300 mg/L cefotaxime and 50 mg/L hygromycin.        Election plates are wrapped with parafilm and placed in the        light at 25° C. The tissue are subcultured onto fresh selection        medium every two weeks. After 6-8 weeks selection the actively        growing callus is distinguished from the brown non-transformed        tissue.

The white proliferating calli in presence of hygromycin are transferredto regeneration medium I (in light at 25° C.). After 2-3 weeks, theregenerated shoot buds are transferred to regeneration medium II (inlight at 25° C.). The rooted plants are transferred to soil and grown ina green house.

Example 5 SAP Gene Expression in Arabidopsis

SAP genes are constitutively expressed in Arabidopsis tissues. FIG. 3shows the PCR amplification of AtSAP11, AtSAP13, AtSAP14, AtSAP12, andAtSAP10, from an Arabidopsis flower cDNA library. FIG. 3 also shows therice OsSAP16 gene product expressed from rice shoot and root cDNAlibraries. The PCR products are resolved on 1% agarose gel.

As shown in FIG. 4, AtSAP genes are induced in response to metal ormetalloid exposure. FIG. 4 shows a semi-quantitative RT-PCR analysis ofArabidopsis AtSAP11, AtSAP13, AtSAP12, and AtSAP10 mRNA expression inroot tissue and rice OsSAP16 mRNA expression in root and shoot tissuesexposed to As(III), As(V), Zn²⁺, and Cd²⁺ at different time intervals(12 and 24 hours) as compared to controls without any metal treatment.ACT2 and ACT1 genes are used as an equal cDNA amount used for RT-PCR andequal loading controls in Arabidopsis and rice, respectively. 250 ngRT-PCR cDNA is used for amplification. PCR cycles numbers and cDNAconcentrations are optimized. Preliminary RT-PCR analysis of mRNAcorresponding to AtSAP11, AtSAP13, AtSAP12 and AtSAP10 genes fromAs(III) and As(V) exposed root tissues show AtSAP11, AtSAP12 and AtSAP10genes are strongly upregulated in response to both As(III) and As(V)after a 12 hour exposure, whereas, AtSAP13 mRNA transcripts are slightlyhigher than controls (FIG. 4). At 24 hours, the transcript levelsdecrease almost to the levels similar to controls in AtSAP11 andAtSAP12. Additionally, the mRNA transcript levels of AtSAP12 at 12 and24 hrs exposure to Zn²⁺ and Cd²⁺ are strongly upregulated in roottissues, slightly upregulated in AtSAP11 and AtSAP13, and mRNA levelsare constitutive in AtSAP10 (FIG. 4). For rice OsSAP16, transcriptlevels are several-fold higher in response to both As(III) and As(V) inroot and shoot tissues (FIG. 4).

As shown in FIG. 5, gene expression of AtSAP13 in transgenic lines isconfirmed by performing semi-quantitative RT-PCR. The mRNA expressionlevels in four AtSAP13 transgenic lines are more than two-fold higher ascompared to wild type (WT) control plants. Actin 2 (ACT2) is used as anequal loading control.

FIG. 6 shows semi-quantitative RT-PCR analysis of AtSAP10 expression inresponse to various stress treatments. Expression analysis is performedwith AtSAP10 specific primers from the RNA isolated from the Arabidopsisseedlings subjected to As(III) and As (V) (A), Cd and Zn (B), Ni (C), Mn(D), ABA (E), Heat (F), Salt (G), and Cold (H) treatments. All upperpanels represent AtSAP10 and lower panel represent ACT2 used as internalloading control. Numbers on each upper panel represents time intervalsin hours for which the stress treatments are given.

Example 6 Germination and Growth

Wild type (Columbia) and transgenic Arabidopsis seeds are sterilized byrinsing in 70% ethanol for 1 minute, then in 30% CLOROX™ bleach (5.25%sodium hypochlorite) for 30 minutes with frequent shaking, followed by 4rinses in sterile water. Sterilized seeds are sown on one half strengthMS medium containing 30 g/liter sucrose, 0.8% PHYTAGAR (purified agar)(GIBCO/BRL, Invitrogen, Carlsbad, Calif.), pH 5.7. The seeds plated onmedia are vernalized at 4° C. for at least 25 hours. Seedlings are grownat 22° C. with a daily regime of 16 hours light/8 hours darkness. Shootsand roots of three-week old individual seedlings are harvestedseparately, rinsed with sterile water, dry-blotted, weighed and rootlength is measured.

Example 7 Metal Resistance of Transgenic Arabidopsis Lines

Arabidopsis AtSAP13 is expressed under the control of a constitutivepromoter (ACT2pt::AtSAP13) in transgenic Arabidopsis plants. Thetransgenic plants are highly resistant to metal concentrations thatinhibit growth in wild type control Arabidopsis. FIG. 7. For metalresistance assays, transgenic plant lines are grown on ½×MS mediasupplemented with different metals. For As(III), As(V), Zn and Cd stresstreatments, wild type Arabidopsis seeds are grown in half strengthliquid MS medium in 250 ml flasks under control conditions (16 hourslight and 8 hours dark at 22° C. and 18° C., respectively) with constantswirling. After 12 days, plants are exposed to toxic metals by addingsodium arsenite at 25 μM, sodium arsenate at 150 μM, cadmium chloride at75 μM, and zinc sulfate at 500 μM. Tissue is collected after 0, 12, and24 hours.

For Ni, Mn, and abscisic acid (ABA) treatments, seeds of wild typeplants are germinated on a nylon mesh placed on half-strength MS agarplates. The 12 days old seedlings along with the supporting mesh aretransferred on a 2 cm long piece of 50 ml Nalgene plastic tube supportplaced in the magenta boxes containing half-strength MS liquid mediumand allowed to acclimatize for additional period of seven days. At theend of acclimatization period, plants are exposed to Ni, Mn, and ABA byadding 90 μM nickel chloride, 1 mM manganese chloride, 154 and 1.5 μMABA, 155 respectively. Without being bound by theory, ABA is aphytohormone thought to regulate plant responses to abioticenvironmental stresses. Shoot and root tissues are harvested separatelyafter 0, 6, 12, and 24 hours.

For heavy metal tolerance/sensitivity analysis, seeds of the wild typeand transgenic plants are germinated and grown on vertically placedhalf-strength MS agar plates (with 1% sucrose) in the absence orpresence of heavy metals for three weeks with a 16 hrs light/8 hrs darkcycle at 22° C./18° C. day/night temperature. To reduce variations, wildtype and AtSAP10 plants are grown side by side on the same plate andtheir growth is compared. Plants are collected, weighed and their rootlengths are measured.

Referring to FIG. 7, constitutive expression of an arsenite-inducibleputative zinc-finger protein (AtSAP13) from a promoter expressioncassette, ACT2pt, confers strong resistance to toxic metals (500micromolar zinc, 25 micromolar arsenite (As(III)), and 75 micromolarcadmium) in Arabidopsis. The T2 homozygous transgenic seeds are grown on½×MS media supplemented with metal concentrations as indicated andplants are allowed to grow for 3 weeks. The transgenic plants have afresh or wet weight that is several fold greater than the wild typeplants and had well-developed, longer roots. As shown in FIGS. 8-10,transgenic AtSAP10 Arabidopsis plants are also resistant to nickel,manganese, and zinc. FIGS. 8-10.

FIG. 8 shows the nickel (Ni) resistance phenotype of Arabidopsis AtSAP10overexpression lines. (A) Ni resistance phenotypes, (B) Fresh shootweight, and (C) root length of three transgenic lines AtSAP10-23,AtSAP10-30, and AtSAP10-42 overexpressing AtSAP10 from ACT2pt expressioncassette and wild type (WT) plants grown on 90 μM NiCl2 in half-strengthMS medium for three weeks. The average and standard deviation (SD)values are represented for four replicates of 12 seedlings each for WTand all AtSAP10 lines. The asterisks represent the significantdifference in biomass accumulation and root length compared with wildtype (WT) plants, (*) P<0.05, (**) P<0.01.

FIG. 9 shows the manganese (Mn) resistance phenotype of ArabidopsisAtSAP10 overexpression lines. (A) Mn resistance phenotypes and (B) Freshshoot weight of three transgenic lines AtSAP10-23, AtSAP10-30, andAtSAP10-42 overexpressing AtSAP10 from ACT2pt expression cassette andwild type (WT) plants grown on 1 mM MnCl₂ in half-strength MS medium 596for three weeks. The average and standard deviation (SD) values arerepresented for four replicates of 12 seedlings each for WT and AtSAP10lines. The asterisks represent the significant difference in biomassaccumulation compared with wild type (WT) plants, (*) P<0.05, (**)P<0.01.

FIG. 10 shows the zinc (Zn) resistance phenotype of Arabidopsis AtSAP10overexpression lines. (A) Zn resistance phenotypes, (B) Fresh shootweight, and (C) root length of three transgenic lines AtSAP10-23,AtSAP10-30, and AtSAP10-42 overexpressing AtSAP10 from ACT2pt expressioncassette and wild type (WT) plants grown on 500 μM ZnSO₄ inhalf-strength MS medium for three weeks. The average and standarddeviation (SD) values are represented for four replicates of 12seedlings each for WT and all AtSAP10 lines. The asterisks represent thesignificant difference in biomass accumulation and root length comparedwith wild type (WT) plants, (*) P<0.05, (**) P<0.01.

These results are unexpected because prior research had demonstratedthat the nematode homologue aip-1 is selectively induced by arsenite andzinc but not other metals. Without being bound by theory, it is believedthat the Cys₂-His₂ zinc finger domains in the plant SAP homologues areable to bind several different metals and metalloids and their ions. Theprotein-metal complexes reduce or eliminate the toxicity of the metal ormetalloid. It is further possible that the protein-metal complexes aresequestered in specialized subcellular compartments.

Example 8 Metal Accumulation in Transgenic Arabidopsis Lines

For heavy metal accumulation analysis, seeds of wild type and transgenicplants are germinated on a nylon mesh placed on half-strength MS agarplates (with 1% sucrose). The 12 days old seedlings along with thesupporting mesh are transferred on a 2 cm long piece of 50 ml Nalgeneplastic tube support placed in the magenta boxes containinghalf-strength MS liquid medium and allowed to acclimate for additionalperiod of seven days. At the end of acclimatization period, thehalf-strength MS liquid medium is replaced 200 with a new liquid mediumcontaining the appropriate amounts of metals and are continued to growfor another four days. At the end of fourth day, plants are removed fromthe magenta boxes, harvested root and shoot separately, washed three tofour times with Mili-Q water and dried in paper folds at 70° C. for 48hours. Dried plant samples are crushed to fine powder, weighed, and thendigested in the concentrated nitric acid (10 mg/ml) with constantshaking for 48 hours. Hydrogen peroxide (30%) is added at the end of 48hrs of acid digestion to promote oxidation of organic matter and achievecomplete digestion. Samples are then centrifuged at 3000 rpm for 10 minand the clear supernatant is diluted 10-fold with deionized water.Samples are analyzed by Elan DRCe inductively coupled plasma-massspectrometry (ICP-MS).

To analyze the uptake of arsenic (As), cadmium (Cd), and zinc (Zn) inthe root and shoot tissues of transgenic plants overexpressing AtSAP13,independent transgenic lines are grown on ½×MS media containing 25millimolar As(III) and 400 millimolar Zn for three weeks. Plant tissuesare harvested, washed, acid digested and metal contents are analyzed byICP-MS. The levels of arsenic in these transgenic lines are similar tothe control plants, whereas, the transgenic lines accumulatedsignificantly higher levels of zinc in both root and shoot tissues ascompared to wild type controls (FIG. 11). The overexpression of AtSAP13gene in transgenic plants caused significant increased accumulation ofzinc in plant tissues without increasing toxic arsenic accumulation.Zinc is a required, but often deficient, nutrient for plants. Therefore,overexpression of the SAP genes in plants and increased zincaccumulation is highly desirable for crop improvement.

FIG. 12 shows the total Ni, Mn, and Zn accumulation in Arabidopsis SAP10overexpression lines. The total Ni concentration in shoots (A) and roots(B) of wild type (WT) and four overexpression lines of AtSAP10 grown onhydroponics medium containing 90 μM NiCl₂. Total Mn accumulation inshoots (C) and roots (D) of wild type (WT) and four overexpressiontransgenic lines of AtSAP10 grown on hydroponics medium containing 1 mMMnCl₂. Total Zn accumulation in shoots (E) and roots (F) of wild type(WT) and four overexpression transgenic lines of AtSAP10 grown onhydroponics medium containing 500 μM ZnSO₄. The average and standarddeviation (SD) values are shown for four replicates of 25 plants eachfor WT and all AtSAP10 lines. Asterisk represents the significantdifference in Ni, Mn, or Zn accumulation as compared to wild type (WT),(*)<0.05, (**)<0.01.

Example 9 Abiotic Stress Resistance of Transgenic Arabidopsis Lines

The AtACR2 transgenic plants are also resistant to salt, drought, andcold stress. Three-week old transgenic plants are grown on 250millimolar NaCl for one week and show strong resistance to salt ascompared to control wild type plants. As shown in FIG. 13, constitutiveexpression of AtSAP13 from a promoter expression cassette, ACT2pt,conferred strong resistance to high salt concentration in Arabidopsis.After the one week salt treatment, the wild type plant died, whereas thetransgenic plants recovered from the salt stress and grew well. Further,these plants are subjected to drought (withholding watering for 8 daysand then recovery with watering) and cold temperature (2° C. for 5days). Preliminary results indicate that AtACR2 plants transgenic plantsshow increased resistance to both stresses.

For heat treatment, magenta boxes containing wild type plants are keptin an incubator at 38° C. for 0.5, 1, 3, and 6 hours. For coldtreatment, magenta boxes with wild type plants are transferred to agrowth chamber maintained at 2° C. for 0.5, 1, 3, and 6 hours. Droughtstress is tested by removing plants from magenta boxes and placing themon a dry paper towel to remove any excess adhered nutrient solution.Plants are air dried by transferring into dry magenta boxes for thedesired time points. Plants are exposed to salt stress by replacingnormal half-strength MS medium with medium supplemented with 150 mMsodium chloride for desired time points. All samples are harvested,washed with deionized water, and stored at −80° C. until further use.

High temperature stress tests are performed by growing wild type andtransgenic plants side by side on half-strength MS agar plates (with 1%sucrose) with a 16-hour light/8-hour dark cycle at 22° C./18° C.light/dark temperatures for 12 days. The seedlings are initially exposedto 38° C. for 90 minutes and then left at room temperature (22° C.) for120 minutes before finally being exposed to 45° C. for 1 hour. All heattreatments are performed in the dark. Plants are allowed to recover in agrowth chamber for 6 days with a 16-hour light/8-hour dark cycle at 22°C./18° C. At the end of the sixth day, plants are again heat shocked at45° C. for 3 hours and then placed in a growth chamber for a 5-dayrecovery period before scoring.

FIG. 14 shows the effect of high temperature stress on wild type (WT)and AtSAP10 overexpression transgenic lines. 12-day old seedlings areheat stressed (HS) as described above. Photographs of representativeplates containing WT and AtSAP10 transgenic lines are taken after 5 daysof recovery.

FIG. 15 shows the effect of drought on wild type (WT) and AtSAP13overexpression transgenic lines. Arabidopsis plants overexpressingAtSAP13 exhibited drought tolerance. Transgenic Arabidopsis plantsoverexpressing AtSAP13 remained healthy, whereas, wild type plants diedunder drought stress. Plants were grown in well-watered soil for 25 daysand then water was withhold for 11 days.

A transgenic plant comprising a recombinant plant SAP coding sequenceoperatively linked to a plant-expressible transcription regulatorysequence will advantageously provide improved environmental stressresistance in plants such as crop plants and other economicallyimportant plants. The environmental stress resistant transgenic plantwill also advantageously increase crop yield. The environmental stressresistant transgenic plant will further provide increased plant biomass.The improved environmental stress resistance, increased crop yield, andincreased plant biomass will also be highly advantageous in biofuelapplications.

The terms “first,” “second,” and the like, “primary,” “secondary,” andthe like, as used herein do not denote any order, quantity, orimportance, but rather are used to distinguish one element from another.The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims.

1. A transgenic plant transformed with an isolated nucleic acidcomprising a plant arsenite-inducible RNA-associated protein codingsequence operatively linked to a plant-expressible transcriptionregulatory sequence, wherein the plant arsenite-inducible RNA-associatedprotein coding sequence encodes a polypeptide that is at least 95%identical to a polypeptide sequence selected from the group consistingof SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, and SEQ ID NO:14, wherein the plant arsenite-inducibleRNA-associated protein coding sequence encodes a polypeptide thatconfers resistance to an environmental stress, wherein greater than orequal to about 25% of transgenic plants are resistant to anenvironmental stress, and wherein the environmental stress inhibits thegrowth of wild type plants.
 2. The transgenic plant of claim 1, whereinthe transgenic plant is selected from the group consisting ofArabidopsis thaliana, canola, sunflower, tobacco, switchgrass,Brachypodium, mustard, crambe, sugar beet, cotton, maize, wheat, barley,rice, sorghum, mangel-wurzels, tomato, mango, peach, apple, pear,strawberry, banana, melon, potato, carrot, lettuce, cabbage, onion,soybean, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa,rye, oats, turf and forage grasses, biofuel, biomass, and bioenergy cropplants, flax, camolina, and oilseed rape, and nut producing plants. 3.The transgenic plant of claim 1, wherein the environmental stress is ametal, metal ion, metalloid, metalloid ion, salinity, drought, cold,heat, submergence, or a combination comprising one of the foregoing. 4.The transgenic plant of claim 1, wherein the transgenic plant isArabidopsis thaliana or rice.
 5. The transgenic plant of claim 1,wherein the plant arsenite-inducible RNA-associated protein codingsequence is derived from Arabidopsis thaliana or rice.
 6. The transgenicplant of claim 1, wherein the plant arsenite-inducible RNA-associatedprotein coding sequence is selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,and SEQ ID NO:7.
 7. The transgenic plant of claim 1, wherein theplant-expressible transcription regulatory sequence comprises aconstitutive promoter, an inducible promoter, a tissue-specificpromoter, an organ-specific promoter, or a combination of one or more ofthe foregoing promoters.
 8. The transgenic plant of claim 7, wherein theconstitutive promoter is a plant ACT2 promoter or a plant ACT1 promoter.9. The transgenic plant of claim 2, wherein the metal, metal ion,metalloid, or metalloid ion is arsenic, arsenate, arsenite, cadmium,chromium, lead, manganese, mercury, nickel, zinc, or a combinationcomprising one of the foregoing.
 10. The transgenic plant of claim 2,wherein the metal, metal ion, metalloid, or metalloid ion is arsenic,arsenate, arsenite, cadmium, manganese, nickel, or zinc.
 11. Thetransgenic plant of claim 1, wherein abscisic acid regulates the plantresponse to the environmental stress.
 12. The transgenic plant of claim1, wherein the transgenic plant has a biomass that is greater than orequal to about 100% of the biomass of a wild type plant.
 13. Thetransgenic plant of claim 1, further comprising an isolated nucleic acidcomprising a phytochelatin biosynthetic enzyme coding sequence that isgreater than or equal to about 95% homologous with a sequence selectedfrom the group consisting of SEQ ID NO:18, SEQ ID NO:19, and SEQ IDNO:20, wherein the phytochelatin biosynthetic enzyme coding sequence hasphytochelatin biosynthesis activity.
 14. A method for producing atransgenic plant that is resistant to an environmental stress comprisingintroducing an isolated nucleic acid comprising an plantarsenite-inducible RNA-associated protein coding sequence that encodes apolypeptide that is at least 95% identical to a polypeptide sequenceselected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14operatively linked to a plant-expressible transcription regulatorysequence into a plant cell or plant tissue; producing a transgenic plantcell or tissue comprising the isolated nucleic acid; and regeneratingthe transgenic plant cell or transgenic plant tissue to provide atransgenic plant that is resistant to an environmental stress, whereingreater than or equal to about 25% of transgenic plants are resistant tothe environmental stress, and wherein the environmental stress inhibitsthe growth of wild type plants.
 15. The method of claim 14, wherein theenvironmental stress is a metal, metal ion, metalloid, metalloid ion,salinity, drought, cold, heat, submergence, or a combination comprisingone of the foregoing.
 16. The method of claim 14, wherein the transgenicplant is Arabidopsis thaliana or rice.
 17. The method of claim 14,wherein the plant-expressible transcription regulatory sequencecomprises a constitutive promoter, an inducible promoter, atissue-specific promoter, an organ-specific promoter, or a combinationof one or more of the foregoing promoters.
 18. The method of claim 14,wherein the plant arsenite-inducible RNA-associated protein codingsequence is selected from the group consisting of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ IDNO:7.
 19. The method of claim 15, wherein the metal, metal ion,metalloid, or metalloid ion is arsenic, arsenate, arsenite, cadmium,chromium, lead, manganese, mercury, nickel, zinc, or a combinationcomprising one of the foregoing.
 20. The method of claim 15, wherein themetal, metal ion, metalloid, or metalloid ion is arsenic, arsenate,arsenite, cadmium, manganese, nickel, or zinc.